Cancer drugs typically produce short-lived clinical remissions due to acquired drug resistance, which can be spontaneously reversible over time. While modeling the acute response to cancer drugs in human tumor cells, we consistently detect a subpopulation of reversibly "drug-tolerant" cells, even from single cell-derived clones. This drug-tolerant phenotype is transiently acquired and relinquished at low frequency by individual cells within the population. The drug-tolerant cancer cells exhibit a distinct chromatin state and are selectively ablated by chromatin-modifying agents, potentially yielding a therapeutic opportunity. Mechanistic insights into this drug- tolerant state will be pursued as follows: AIM 1. To establish the mechanism by which the subpopulation of drug-tolerant cancer cells maintain viability upon lethal exposure. We will test a hypothesis, based on preliminary findings, that IGFR signaling maintains the drug-tolerant population. We will also consider potential roles for other cellular proteins or pathways in drug tolerance, making use of genomic and proteomic technologies to compare the expression and activation of proteins and pathways in drug-sensitive and drug- tolerant populations. Some of the identified candidate mediators of drug tolerance will be functionally validated using shRNA technology, and these could constitute novel therapeutic targets for the development of inhibitors that prevent drug resistance. AIM 2. To define the basis for phenotypic heterogeneity in a clonally-derived cancer cell population that yields the drug-tolerant subpopulation. Here we will test a hyothesis that epithelial- mesenchymal transformation (EMT) is critical in the establishment of drug tolerance. By sorting epithelial and mesenchymal populations, we will be able to directly test the role of EMT in drug tolerance. We will also examine the relationship between drug-tolerant cells and cancer stem cells. AIM 3. To determine the basis for hypersensitivity to chromatin-modifying agents within the drug-tolerant sub-population. Here, we will examine a role for specific HDACs in drug tolerance and we will test a hypothesis that sensitivity to chromatin modification in the drug-tolerant cells reflects an altered DNA damage response. Acquired drug resistance remains a key limitation to successful cancer therapy. Further understanding of the mechanistic basis for the drug-tolerant state that we have identified could have important implications for the clinical management, and possibly prevention of resistance to cancer drugs. PUBLIC HEALTH RELEVANCE: Narrative Cancer is a leading cause of death worldwide, and the development of drug resistance during treatment with anti-cancer agents remains a critical problem that limits the clinical benefit of cancer drug therapy. We have discovered a novel cellular state-referred to as the "drug-tolerant" state-that appears to contribute to the development of resistance of tumor cells to a variety of cancer drugs. By studying the mechanisms that underlie this state, we expect to be able to develop effective therapeutic strategies to disrupt drug tolerance, thereby improving the efficacy of cancer drugs.